712 Journal of Health Science, 56(6) 712–716 (2010)
— Research Letter —
Safety Evaluation of Black Rice the present study suggests that kurosu from a jar does
not have toxic effect and does not alter expression lev-
Vinegar (Kurosu) from a Jar on els of principal drug metabolism enzymes and trans-
Food-drug Interaction: 30-day porters.
Ingestion Study on Expressions
Key words —— black rice vinegar, cytochrome P450,
of Drug Metabolism Enzymes and transporter
Transporters in Rats
Yoshihiko Shibayama,∗, a Masanobu Nagano, b INTRODUCTION
Akira Fujii, b Miyuki Taguchi, c Yasuo Takeda, c
Food-drug interaction evidence has been accu-
and Katsushi Yamadac
mulated; natural products can interact with drugs
a
Education Research Center for Clinical Pharmacy, Faculty of by the same mechanisms as drugs. Clinically im-
Pharmaceutical Sciences, Hokkaido University, Nishi 6, Kita portant interactions appear to involve effects on
12, Kita-ku, Sapporo 060–0812, Japan, b Sakamoto Brewing drug metabolism via cytochrome P450 (Cyp) isoen-
Co., Ltd., 21–15 Uenosnono-cho, Kagoshima 890–0052, Japan zymes, impairment of hepatic or renal function,
and c Clinical Pharmacy and Pharmacology, Graduate School and other possible mechanisms.1) For example, a
of Medical and Dental Sciences, Kagoshima University, 8–35– healthcare supplement St. John’s Wort, interacts
1 Sakuragaoka, Kagoshima 890–8520, Japan with prescription drugs.1, 2) The family of ATP bind-
(Received February 18, 2010; Accepted August 17, 2010; ing cassette (ABC) transporters, for example, p-
Published online August 23, 2010) glycoprotein (P-gp), multidrug resistance protein 2
(Mrp2) and breast cancer resistance protein (Bcrp),
Black rice vinegar (kurosu) made from a jar is a also has important roles in the detoxification and
traditional vinegar in Japan. Kurosu has been com- excretion of xenobiotics.3) In hepatocytes, organic
monly used as a healthcare supplement; however, it anion transport polypeptide 2 (Oatp2) plays impor-
is not clear whether kurosu has a treatment effect tant roles in the vector transport of bile acids from
on the expression of drug metabolism enzymes and blood to bile. Bile acid is a regulation factor of hep-
transporters, whose expression alterations may in- atic and intestinal transporters.4) The organic anion
duce food-drug interaction. Alteration of the prin- transporter (Oat) family also plays important roles
cipal drug metabolism enzymes and expression fol- in the elimination of a variety of endogenous sub-
lowing kurosu and moromi, the residue from black stances, xenobiotics and their metabolites from the
vinegar brewing, for 30-day treatment was evaluated. body.5)
Water, 0.23% acetic acid, concentrated kurosu (con- Black rice vinegar (kurosu) from a jar is a tradi-
taining 0.23% acetic acid) and moromi was admin- tional vinegar in Kagoshima, Japan.6) It has been re-
istered to Wister rats. The treatment did not signif- ported that kurosu improved hypertension, allergy,
icantly affect the biochemical parameters of serum, hypercholesterol, carbohydrate metabolism and tu-
alanine aminotransferase, alkaline phosphatase, to- mor growth.6, 7) Kurosu has been commonly used
tal protein, creatinine in the serum, and body weight. as a healthcare supplement; however, it is not clear
The treatment also did not affect the expression lev- whether kurosu treatment effects on the expression
els of cytochrome P450 (Cyp) 1a2, Cyp2b1/2, Cyp3a1, of drug metabolism enzymes and transporters.
glutathione S-transferase, p-glycoprotein, multidrug To evaluate the food-drug interaction with
resistance protein 2, breast cancer resistance protein, kurosu, the authors investigated the effect of kurosu
organic anion transport polypeptide 2, and organic treatment on the expression of principal drug
anion transport 2 and 3 in the liver. In conclusion, metabolism enzymes and transporters.
∗
To whom correspondence should be addressed: Education Re-
search Center for Clinical Pharmacy, Faculty of Pharmaceutical
Sciences, Hokkaido University, Nishi 6, Kita 12, Kita-ku, Sap- MATERIALS AND METHODS
poro 060–0812, Japan. Tel. & Fax: +81-11-706-3705; E-mail:
sibayama@pharm.hokudai.ac.jp Chemicals —— Primary antibodies for Western
C 2010 The Pharmaceutical Society of Japan
No. 6 713
Table 1. Primary Antibody Properties and Immunodetection Condition
Molecular Clone or catalog No. Host Protein Dilution ratio
size (kDa) (µg) (primary antibody)
β-actin 43 SIGMA-ALDRICH, AC-74 Mouse 10 10,000
Cyp1a2 58 MILLIPORE, AB10089 Rabbit 20 2,000
Cyp2b1/2 56 BIOMOL, CR 3290 Rabbit 20 1,000
Cyp3a1 58 BIOMOL, CR 3310 Rabbit 10 3,000
GSTa) 26 ROCKLAND, 3D4 Mouse 10 2,000
Mrp2 170 MONOSAN, M2 III-6 Mouse 20 400
P-gp 160 Calbiochem, C219 Mouse 20 1,000
Bcrp 60 Kamiya Biomedical, bxp-21 Mouse 20 500
Oatp2 90 Santa Cruz, sc-33610 Rabbit 20 500
Oat2 60 TransGenic, KE031 Rabbit 50 250
Oat3 130 TransGenic, KE035 Rabbit 50 250
Whole cell homogenate of the liver was separated with SDS-PAGE. The amount of protein and the dilution ratio of the
primary antibodies are indicated. Clone name of mouse monoclonal antibodies or catalog number of polyclonal rabbit antibody
is shown in the “Clone or catalog No.” lane. a) GST: glutathione s-transterase.
blotting detection were purchased from Sigma- treated by oral gavage once a day as follows: ve-
Aldrich Japan (Tokyo, Japan), Nihon Millipore hicle group, distilled water was given at a dose of
K.K. (Tokyo, Japan), BIOMOL (Plymouth Meet- 10 ml/kg; acetic acid group, 1.5 ml of 0.23% acetic
ing, PA, U.S.A.), ROCKLAND (Gilbertsville, PA, acid (pH 3.5) was diluted with 8.5 ml distilled wa-
U.S.A.), MONOSAN (AM Uden, The Nether- ter, and the diluted solution was given at a dose
lands), Calbiochem (San Diego, CA, U.S.A.), of 10 ml/kg; black vinegar group, 1.5 ml concen-
Kamiya Biomedical (Seattle, WA, U.S.A.), Santa trated black vinegar solution was diluted with 8.5 ml
Cruz Biotechnology (Santa Cruz, CA, U.S.A.) and distilled water, and the diluted solution was given
TransGenic (Kumamoto, Japan). The clone names at a dose of 10 ml/kg; moromi group, 50 mg mo-
and catalog numbers of the primary antibodies romi was suspended in 10 ml distilled water, and the
are summarized Table 1. The second antibod- suspension was given at a dose of 10 ml/kg. The
ies, anti-rabbit and anti-mouse IgG antibody con- rats were killed by exsanguination after blood col-
jugated with horseradish peroxidase (HRP), were lection. As a positive control treatment, dexam-
purchased from Nacalai Tesque (Kyoto, Japan). ethasone was intraperitoneally injected at a dose of
Nitrocellulose membranes, ECL Western blotting 50 mg/kg for consecutive 4 days8, 9) and methotrex-
detection system and Hyperfilm ECL were pur- ate was intraperitoneally injected once at a dose of
chased from GE Healthcare U.K. Ltd. (Bucking- 150 mg/kg.10, 11) Dexamethasone-treated rats were
hamshire, U.K.). Protease inhibitor cocktail tablets killed the day after 4-day treatment. Methotraxate-
(Complete , EDTA free) were purchased from
R
treated rats were killed on day 4 after treatment.
Roche Diagnostics GmbH (Basel, Switzerland). All Target tissues were promptly removed and stored
other reagents were purchased from Wako Pure at −30◦ C until Western blot analysis. Serum pre-
Chemical Industries (Osaka, Japan). Concentrated pared from each rat was stored at 4◦ C until analysis.
black vinegar from a jar (10-fold concentrated, pH. Hemolysate samples were excluded from samples
4.41, acetic acid distilled) and moromi (residue for analysis. Animal experiments were performed
from black vinegar brewing) were obtained from in accordance with the criteria of Kagoshima Uni-
Sakamoto Brewing Co., Ltd. (Kagoshima, Japan). versity for the care and use of experimental animals.
Concentrated black vinegar contains 0.23% acetic Assays —— Creatinine, alanine aminotransferase
acid, 3.23% lactic acid and 1.99% ash. (ALT) and alkaline phosphatase (ALP) were de-
Animals —— Male Wistar rats (body weight, termined with the clinical reagents for diagnosis
90 ± 5 g, mean ± S.D., Kyudo, Kumamoto, Japan) (Shino-test, Tokyo, Japan). Protein concentra-
were housed under standard conditions (21 ± 2◦ C, tion was determined by DC protein assay (Bio-
ventilated rooms, 12 hr light/dark cycle). The ani- Rad Japan, Tokyo, Japan). Optical densities (ODs)
mals were fed rat chow and allowed free access to were measured by the public domain program Im-
water during the experimental period. Rats were age J (developed by the U.S. National Institutes
714 Vol. 56 (2010)
of Health and available on the Internet at http:// further incubated with 1000-fold diluted secondary
rsb.info.nih.gov/ij/download.html). Individual ex- antibody conjugated with HRP. Finally, membranes
posures were scanned into TIFF images with a were rinsed once for 15 minutes and four more
GT9000 color scanner (Seiko Epson, Tokyo, Japan). times for 5 minutes with buffer A. For detection,
Overall differences among treatments were evalu- the ECL Western blotting detection system was
ated by one-way analysis of variance (ANOVA). used.
Differences among groups in vehicle-, acetic acid-,
black vinegar- and moromi-treated rats were evalu-
ated using ANOVA. RESULTS AND DISCUSSION
Western Blotting —— Whole cell homogenate
was prepared from the rat liver. Tissues ob- Rats were administered water, acetic acid, black
tained from individual animals of each group vinegar and moromi once a day for 30 consecutive
were cryopreserved and homogenized in buffer days. It was reported that the dose of black vinegar
containing 250 mM sucrose and 5 mM N-2- and moromi corresponded to the human dose and
hydroxyethylpiperazine-N -2-ethanesulfonic acid that black vinegar treatment improved blood sugar
(HEPES)-tris(hydroxymethyl)-aminomethane (Tris, levels in a diabetic model mice.6) The biochemi-
pH 7.4) with an ultrasonic homogenizer. Protease cal parameters of serum, ALT, ALP, total protein,
inhibitor tablets were dissolved in the above- creatinine were measured to estimate general liver
mentioned homogenizing buffer at appropriate and kidney functions. Acetic acid, black vinegar
concentrations. Protein samples were mixed with and moromi treatments showed no significant differ-
an equal volume of sodium dodecyl sulfate (SDS) ences in the biochemical parameters of serum and
sample buffer containing 125 mM Tris-HCl (pH body weight between water treatments (Table 2).
6.8), 4% SDS, 20% glycerol and 0.005% bro- This result suggests that acetic acid, black vinegar
mophenol blue. Protein samples for P-gp, Mrp2, and moromi treatment have no toxic effect.
Bcrp, Oat2 and Oat3 were not heat treated.12) An example of Western blot analysis is shown
Western blotting for P-gp, Mrp2 and Oat3 detection in Fig. 1. β-actin, Cyp2b1/2, Cyp3a1, P-gp, Mrp2
was separated by 7.5% SDS-polyacrylamide gel and Bcrp were detected as 43, 56, 58, 160, 170
electrophoresis (PAGE). The other protein samples and 60 kDa bands, respectively. As a positive con-
were separated by 12% SDS-PAGE. Western blot- trol treatment for up-regulation of Cyp2b1/2 and
ting and primary antibody dilution are summarized Cyp3a1, dexamethasone treatment increased the ex-
in Table 1. After SDS-PAGE separation, proteins pression levels of Cyp2b1/2 and Cyp3a1, but the
were transferred to a nitrocellulose membrane. treatment reduced expression levels of P-gp and
After transfer, the membranes were incubated with Bcrp.8, 9) As a positive control treatment for down-
3% skim milk in buffer A (0.35 M NaCl, 10 mM regulation of P-gp, Mrp2 and Bcrp, methotrexate
Tris-HCl, pH 8.0, 0.05% Tween 20) for 1 hr at treatment also reduced expression levels of P-gp,
room temperature for blocking. The membranes Mrp2 and Bcrp (Fig. 1).10, 11) Expression levels of
were then incubated with primary antibody under the protein are described as a percentage of the cor-
the same conditions as for blocking. After washing responding water-treated group (Table 3). Acetic
three times with buffer A, the membranes were acid, black vinegar and moromi treatments showed
Table 2. Biochemical Parameters of Serum and Body Weight after 30 Consecutive Days Treatment in Rats
Water Acetic acid Black vinegar Moromi
Body weight (g) 359.8 ± 6.4 343.2 ± 6.8 341.3 ± 10.6 342.5 ± 4.6
Total protein (g/dl) 3.35 ± 0.30 3.22 ± 1.08 3.21 ± 0.41 3.49 ± 0.78
Creatinine (mg/dl) 0.69 ± 0.13 0.85 ± 0.23 0.56 ± 0.18 1.05 ± 0.24
ALP (IU/l) 264 ± 38 307 ± 34 355 ± 46 355 ± 39
ALT (IU/l) 30.5 ± 4.5 25.4 ± 3.3 24.7 ± 2.5 18.3 ± 1.3
Each group was treated as follows: water, distilled water was given at a dose of 10 ml/kg; acetic acid, 1.5 ml of 0.23%
acetic acid (pH 3.5) was diluted with 8.5 ml distilled water, and the diluted solution was given at a dose of 10 ml/kg;
black vinegar, 1.5 ml concentrated black vinegar solution (pH 3.5) was diluted with 8.5 ml distilled water, and the diluted
solution was given at a dose of 10 ml/kg; moromi, 50 mg moromi suspended in 10 ml distilled water, and the suspension
was given at a dose of 10 ml/kg. Data are the means ± S.E. (N = 6).
No. 6 715
Table 3. Relative Expression Levels of the Drug Metabolism Enzymes and Transporters
after Kurosu Treatment with Consecutive 30-days
Water Acetic acid Black vinegar Moromi
Actin 100.0 ± 5.1 98.5 ± 7.3 101.5 ± 9.1 116.4 ± 7.3
Cyp1a2 100.0 ± 10.1 116.1 ± 12.5 89.3 ± 9.5 102.6 ± 8.1
Cyp2b1 100.0 ± 4.5 112.9 ± 4.2 112.7 ± 6.2 95.5 ± 2.9
Cyp3a1 100.0 ± 11.8 81.1 ± 7.9 85.8 ± 5.2 89.4 ± 7.0
GST 100.0 ± 16.1 97.3 ± 7.2 108.2 ± 22.5 118.7 ± 12.2
P-gp 100.0 ± 7.7 81.9 ± 6.1 82.7 ± 7.2 79.7 ± 9.6
Mrp2 100.0 ± 7.5 99.1 ± 2.5 85.7 ± 2.5 78.9 ± 3.2
Bcrp 100.0 ± 9.9 90.1 ± 12.6 90.8 ± 6.0 77.2 ± 11.4
Oatp2 100.0 ± 6.8 85.2 ± 6.4 93.7 ± 9.2 83.4 ± 7.2
Oat2 100.0 ± 9.6 80.8 ± 3.1 79.3 ± 10.5 87.7 ± 11.4
Oat3 100.0 ± 7.8 85.2 ± 15.4 90.0 ± 8.0 113.2 ± 22.7
Rats were killed after 30-day treatment, and the preparation of samples for Western blot anal-
ysis was similar to that described in Fig. 1. Each group was treated as follows: water, distilled
water was given at a dose of 10 ml/kg; acetic acid, 1.5 ml of 0.23% acetic acid (pH 3.5) was diluted
with 8.5 ml distilled water, and the diluted solution was given at a dose of 10 ml/kg; black vinegar,
1.5 ml concentrated black vinegar solution (pH 3.5) was diluted with 8.5 ml distilled water, and
the diluted solution was given at a dose of 10 ml/kg; moromi, 50 mg moromi suspended in 10 ml
distilled water, and the suspension was given at a dose of 10 ml/kg. Values are the mean ± S.E.
(percentage) of normalized ODs, compared with vehicles, N = 6. There were no significant differ-
ences in expression levels. Statistics analysis was performed by ANOVA.
Oguma et al. reported that combined admin-
istration of kurosu increased maximum concentra-
tion of itraconazole in healthy adults with decrease
gastric acidity and showed high blood concentra-
tion of itraconazole in a pulmonary aspergillosis pa-
tient.13) Iwao et al. reported that physicochemical
membrane permeability of glibenclamide was re-
duced in the presence of kurosu.14) These reports
demonstrated that acidic physicochemical property
of kurosu may affect absorption of drugs. However,
it is not clear whether kurosu induce drug interac-
tion through alteration of drug metabolism enzymes
and transporters.
In conclusion, the present study suggest that
Fig. 1. Effect of Kurosu and Moromi Treatment on Expression
kurosu from a jar has no toxic effect and does
Levels of β-actin, Cyp2b1/2, Cyp3a1, P-gp, Mrp2 and
Bcrp not alter the expression levels of the principal drug
Rats were gavaged orally for consecutive 30 days. β-actin, metabolism enzymes and transporters.
Cyp2b1/2, Cyp3a1, P-gp, Mrp2 and Bcrp were detected as 43, 56, 58,
160, 170 and 60 kDa bands, respectively. As a positive control treat-
ment for up-regulation of Cyp2b1/2 and Cyp3a1, dexamethasone was Acknowledgement This study was supported in
intraperitoneally injected at a dose of 50 mg/kg for consecutive 4 days. part by a Grant-in-aid from the Japan Society for
As a positive control treatment for down-regulation of P-gp, Mrp2 and the Promotion of Science (No. 20926005).
Bcrp, methotrexate was intraperitoneally injected once at a dose of
150 mg/kg. Whole-cell homogenate from the liver was prepared and
loaded onto SDS-PAGE. The detailed procedure for Western blot anal-
ysis is described in ‘Materials and Methods.’ AcOH: 0.23% acetic acid,
B.V.: black vinegar, DEX: dexametasone, MTX: methotrexate.
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